Increased production of ginsenosides through improvement of protein-folding machinery of yeast

Abstract

The present invention relates to recombinant yeast, in which the productivity of ginsenoside is enhanced by overexpressing CPR5, PDI1, or ERO1 in yeast having the productivity of ginsenosides; a method for preparing the yeast; and a method for producing ginsenosides using the yeast.

Claims

1. A recombinant yeast for producing a ginsenoside or a precursor thereof, wherein the recombinant yeast expresses a polynucleotide that encodes a protein involved in protein folding having at least 95% sequence homology to SEQ ID NO:1 or SEQ ID NO:3, wherein the protein involved in protein folding has Cyclosporin-sensitive Proline Rotamase 5 (CPR5)-like activity, or ER Oxidation or Endoplasmic Reticulum Oxidoreductin (ERO1)-like activity; wherein the recombinant yeast has elevated expression levels of the protein involved in protein folding in comparison to endogenous expression levels of the protein; and wherein the recombinant yeast comprises ginsenoside synthesizing genes encoding the proteins of HMG-CoA reductase (tHMG1), Panax ginseng squalene epoxidase (PgSE), Panax ginseng dammarenediol-ll synthase (PgDDS), Panax ginseng cytochrome P450 CYP716A47 (PgPPDS), and Panax ginseng NADPH-cytochrome P450 reductase (PgCPR).

2. The recombinant yeast of claim 1, wherein the polynucleotide has at least 99% sequence homology to SEQ ID NO:1 and the protein involved in protein folding has CPR5-like activity.

3. The recombinant yeast of claim 1, wherein the polynucleotide has at least 99% sequence homology to SEQ ID NO:3 and the protein involved in protein folding has ERO1-like activity.

4. The recombinant yeast of claim 1, wherein the yeast is selected from the group consisting of S. cerevisiae, S. bayanus, S. boulardii, S. bulderi, S. cariocanus, S. cariocus, S. chevalieri, S. dairenensis, S. ellipsoideus, S. eubayanus, S. exiguus, S. florentinus, S. kluyveri, S. martiniae, S. monacensis, S. norbensis, S. paradoxus, S. pastorianus, S. spencerorum, S. turicensis, S. unisporus, S. uvarum, and S. zonatus.

5. The recombinant yeast of claim 1, wherein the expression levels of tHMG1, PgSE, PgDDS, PgPPDS, and PgCPR are elevated in comparison to their endogenous expression levels.

6. The recombinant yeast of claim 1, wherein the precursor is squalene or 2,3-oxidosqualene.

7. The recombinant yeast of claim 1, wherein a vector comprises the polynucleotide and expression of the polynucleotide is driven by a PGK1 promoter.

8. The recombinant yeast of claim 1, wherein the recombinant yeast comprises ginsenoside synthesizing genes encoding proteins having the sequence of SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:8.

9. The recombinant yeast of claim 4, wherein the recombinant yeast is S. cerevisiae.

10. The recombinant yeast of claim 9, wherein the recombinant yeast has been recombinantly modified to express tHMG1, PgSE, PgDDS, PgPPDS, and PgCPR proteins from vector(s) comprising ginsenoside synthesizing genes.

11. The recombinant yeast of claim 10, wherein the expression of the ginsenoside synthesizing genes is driven by a Glycerol-3-Phosphate Dehydrogenase (GPD1) promoter.

12. The recombinant yeast of claim 11, wherein expression of the polynucleotide results in about a 5-fold increase in production of the ginsenoside in comparison to non-expression of the polynucleotide.

13. A method for preparing recombinant yeast with an enhanced productivity of ginsenosides compared to parent yeast, comprising transforming the recombinant yeast cell with a polynucleotide having the sequence of SEQ ID NO:1 and/or SEQ ID NO:3 such that expression of a protein encoded by the polynucleotide is increased, and wherein the protein has CPR5-like activity or ERO1-like activity in a ginsenoside-producing yeast strain relative to its endogenous expression levels.

14. The method of claim 13, wherein the ginsenoside-producing yeast strain is one or more selected from the group consisting of Panax ginseng dammarenediol-ll synthase (PgDDS), Panax ginseng cytochrome P450 CYP716A47 (PgPPDS), Panax ginseng NADPH-cytochrome P450 reductase (PgCPR), S. cerevisiae HMG-CoA reductase (tHMG1), and Panax ginseng squalene epoxidase (PgSE).

15. A method for preparing recombinant yeast with an enhanced productivity of ginsenoside precursors compared to parent yeast, comprising increasing the expression level of a protein encoded by the sequence of SEQ ID NO:1 and/or 3, and wherein the protein has CPR5-like activity or ERO1-like activity in a ginsenoside precursor-producing yeast strain relative to its endogenous expression levels.

16. The method of claim 15, wherein the ginsenoside precursor comprises squalene or 2,3-oxidosqualene.

17. A method for producing ginsenoside or a precursor thereof, comprising culturing the recombinant yeast of any one of claim 1.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 is a simplified illustration of an increase in the production amount of ginsenosides through improved protein folding.

(2) FIG. 2 is diagram showing a metabolic pathway of ginsenoside biosynthesis.

(3) FIG. 3 is a diagram showing a vector map of pUC57-URA3HA-PGK1, which is a vector prepared for overexpressing the CPR5, PDI1, or ERO1 gene.

(4) FIG. 4 is a graph showing the amounts of squalene, 2,3-oxidosqualene, and protopanaxadiol productions in comparison with a control group, when CPR5, PDI1, or ERO1 are overexpressed.

DETAILED DESCRIPTION

(5) Hereinbelow, the present invention will be described in detail with accompanying exemplary embodiments. However, the exemplary embodiments disclosed herein are only for illustrative purposes and should not be construed as limiting the scope of the present invention.

Example 1: Construct of PPD Modified Yeast Strain

(6) In S. cerevisiae CEN.PK2-1D wild-type strains [(MATα ura3-52; trp1-289; leu2-3,112; his3Δ1; MAL2-8; SUC2), EUROSCARF accession number: 30000B], the metabolic pathway of ginsenoside biosynthesis was introduced and the metabolic pathway of mevalonic acid for enhancing the biosynthesis of squalene was enhanced, which is a precursor essential for ginsenoside biosynthesis. Thereafter, the yeast strain producing protopanaxadiol (PPD) was constructed, and this yeast strain was named as a modified PPD yeast strain.

(7) The genotype of the PPD strain is S. cerevisiae CEN.PK2-1D Δtrp1::P.sub.GPD1 tHMG1+P.sub.GPD1 PgSE+Δleu2::P.sub.GPD1 PgDDS+P.sub.GPD1 PgPPDS+P.sub.GPD1 PgCPR. Genes encoding Panax ginseng dammarenediol-II synthase (PgDDS; SEQ ID NO: 4), Panax ginseng cytochrome P450 CYP716A47 (PgPPDS; SEQ ID NO: 5), and Panax ginseng NADPH-cytochrome P450 reductase (PgCPR; SEQ ID NO: 6), which are ginsenoside biosynthetic enzymes, and genes encoding S. cerevisiae HMG-CoA reductase (tHMG1; SEQ ID NO: 7) and Panax ginseng squalene epoxidase (PgSE; SEQ ID NO: 8), which are enzymes for enhancing the metabolic pathway of mevalonic acid, were each transcribed from the GPD1(TDH3) promoter, which is a strong constitutive promoter, so as to be expressed.

Example 2: Construct of CPR5-, PDI1-, or ERO1-Overexpressing Modified Yeast Strain

(8) In the modified PPD yeast strain, in order to confirm whether overexpression of CPR5, PDI1, or ERO1, which are proteins involved in protein folding, is involved in the growth of the modified yeast strain and the PPD-producing ability, the modified yeast strain was constructed in which CPR5, PDI1, or ERO1 gene is overexpressed. First, in order to induce overexpression of the CPR5, PDI1, or ERO1 gene, a PGK1 promoter replacement vector was constructed for substituting the promoter of the gene to a PGK1 promoter, which is a strong constitutive promoter, and then the substitution cassette constructed using the vector was transfected into the modified PPD yeast strain, and thereby a modified yeast strain overexpressing CPR5, PDI1, or ERO1 was constructed.

(9) Specifically, in order to construct the PGK1 promoter replacement vector, target fragments were obtained by PCR amplification from the genomic DNA of S. cerevisiae CEN.PK2-1 using primers in a combination of PGK1 pro F and PGK1 pro R primers (Table 1), such that the sequence of a site of the PGK1 promoter (i.e., a strong constitutive promoter) has recognition sites for restriction enzymes SacI and XbaI at the 5′ and 3′ sites of the promoter, respectively, followed by conducting electrophoresis of the amplified PCR fragments. Herein, the PCR was carried out for a total of 25 cycles under the following conditions: denaturation at 95° C. for 30 seconds, annealing at 56° C. for 30 seconds, and elongation at 72° C. for 30 seconds. The amplified fragments were treated with SacI and XbaI, and then inserted into a pUC57-URA3HA vector treated with the same restriction enzymes. Engineering cellular redox balance in Saccharomyces cerevisiae for improved production of L-lactic acid. Biotechnol. Bioeng., 112, 751-758.), thereby constructing a pUC57-URA3HA-PGK1 vector (SEQ ID NO: 9) (FIG. 3).

(10) The primer sequences and restriction enzymes used for constructing the pUC57-URA3HA-PGK1 vector are shown in Table 1 below.

(11) TABLE-US-00001 TABLE 1 SEQ ID Restriction Primer Primer Sequence NO Enzyme PGK1 pro F 5′-CGAGCTCAGACGCGAATTTTTCGGG-3′ 10 SacI PGK1 pro R 5′-GACTAGTTCTAGATGTTTTATATTTGTTGTAAAA 11 XbaI AGTAGATAATTACTTCC-3′

(12) PCR was carried out with primers in a combination of P_CPR5 F and P_CPR5 R (SEQ ID NOS: 12 and 13), which have the homologous recombination sequences to the CPR5 promoter sites, using the thus-prepared pUC57-URA3HA-PGK1 vector as a template. Similarly, the cassette substituting the CPR5 promoter with the PGK1 promoter was constructed. In addition, PCR was carried out with primers in a combination of P PDT′ F and P_PDI1 R (SEQ ID NOS: 14 and 15), which have the homologous combination sequences to the PDI1 promoter sites, to construct the cassette substituting the PDI1 promoter with the PGK1 promoter. In addition, PCR was carried out with primers in a combination of P_ERO1 F and P_ERO1 R (SEQ ID NOS: 16 and 17), which have the homologous sequences to the ERO1 promoter sites, to construct the cassette constituting the ERO1 promoter with the PGK1 promoter. Herein, the PCR was carried out for a total of 25 cycles under the following conditions: denaturation at 95° C. for 30 seconds, annealing at 56° C. for 30 seconds, and elongation at 72° C. for 2 minutes.

(13) The thus-prepared cassette for substituting the CPR5 promoter, PDI1 promoter, or ERO1 promoter was each introduced into the modified PPD yeast strain. The introduction was carried out by a common heat shock transformation. After the transformation, cells were cultured in uracil dropout medium (yeast nitrogen base without amino acids 6.7 g, CSM minus uracil 0.77 g, glucose 20 g, 1 L), and the CPR5 promoter, PDI1 promoter, or ERO1 promoter on the genome was allowed to be substituted with the PGK1 promoter by each of the cassettes above.

(14) In order to confirm whether each of the promoters was substituted with the PGK1 promoter in the thus-obtained modified yeast strain, PCR was carried out with primers in a combination of CPR5 to PGK1 F and CPR5 to PGK1 R (SEQ ID NOS: 18 and 19) using the genome of the cells above as a template; as a result, it was confirmed that CPR5 promoter was substituted with the PGK1 promoter. In addition, PCR was carried out with primers in a combination of PDI1 to PGK1 F and PDI1 to PGK1 R (SEQ ID NOS: 20 and 21) or a combination of ERO1 to PGK1 F and ERO1 to PGK1 R (SEQ ID NOS: 22 and 23), and as a result, it was confirmed that the PDI1 promoter or the ERO1 promoter was substituted with the PGK1 promoter. Based on the results above, PPD-CPR5(P.sub.CPR5::P.sub.PGK1), PPD-PDI1(P.sub.PDI1::P.sub.PGK1), and PPD-ERO1(P.sub.ERO1::P.sub.PGK1) modified yeast strains were prepared.

(15) The primer sequences for preparing the PGK1 promoter substitution cassette and for confirming the substitution are shown in Table 2 below.

(16) TABLE-US-00002 TABLE 2 SEQ ID Primer Primer Sequence NO P_CPR5 F 5′-ACTAGAAGAATTTGTATCTTCTGATCCTGGTTTAACACA 12 ATGGTTATAGTAGGTTTCCCGACTGGAAAGC-3′ P_CPR5 R 5′-GTGAAGAGACAAGCAAATAAGGTAATAAAGGAAAAAAAT 13 TGAAGCTTCATTGTTTTATATTTGTTGTAGTAGATAA-3′ P_PDI1 F 5′-CTTATAATGCGGGGTGCAAGCGCCGCGTCTAAAATTTTT 14 TTTTTTTCCATAGGTTTCCCGACTGGAAAGC-3′ P_PDI1 R 5′-GCGAGCAGCAGGGAGGACCATGACAGGACGGCACCAGCA 15 GAAAACTTcatTGTTTTATATTTGTTGTAGTAGATAA-3′ P_ERO1 F 5′-GTAAAATTGTACATTATTTATTTCTATATAACAGG 16 ATCCCTCCAGTAGGTTTCCCGACTGGAAAGC-3′ P_ERO1 R 5′-GATGTAAAAGCCGTGAGGCACAGTGTGGCAATGGCGGT 17 TCTTAATCTCATTGTTTTATATTTGTTGTAGTAGATAA-3′ CPR5 to PGK1 F 5′-TCTTCTGATCCTGGTTTAACACAATGG-3′ 18 CPR5 to PGK1 R 5′-CTTTCGCTGGCTGTTGTGAA-3′ 19 PDI1 to PGK1 F 5′-AAGCGCCGCGTCTTTT-3′ 20 PDI1 to PGK1 R 5′-GGGAGGACCATGACAGGACG-3′ 21 ERO1 to PGK1 F 5′-GTGCTGTGTACACCCGTAAAATTGT-3′ 22 ERO1 to PGK1 R 5′-GAGGCACAGTGTGGCAATGG-3′ 23

Example 3: Confirmation of Growth of Transformed, Modified Yeast Strain and Amount of PPD Production

(17) The transformed, modified yeast strains prepared above were inoculated into minimal URA drop-out media (50 mL) containing 2% glucose such that the OD.sub.600 became 0.5. Thereafter, the resultants were cultured under aerobic conditions for 144 hours while stirring at 30° C. at 250 rpm. The OD.sub.600 value of the cell growth during the culture was measured using a spectrophotometer. The intracellular metabolites (e.g., squalene, 2,3-oxidosqualene, and protopanaxadiol) during the culture were analyzed using high-performance liquid chromatography (HPLC).

(18) As a result of culturing for 72 hours and 144 hours, the cell growths, i.e., the OD.sub.600 value of the culture and the concentrations of each intracellular metabolite, are as shown in Tables 3, 4, and FIG. 4.

(19) The concentrations of the metabolites according to the culture of the transformed, modified yeast strains prepared above are shown in Table 3 below.

(20) TABLE-US-00003 TABLE 3 Cell Growth Amount of Metabolite Production (mg/L) (0D.sub.600) Squalene 2,3-oxidosqualene Protopanaxadiol Strain 72 h 144 h 72 h 144 h 72 h 144 h 72 h 144 h Control 17.44 15.69 0.19 1.13 0.64 0.69 0.25 1.67 CPR5-overexpressing strain 18.65 15.86 0.96 1.67 0.87 0.41 1.35 8.25 PDI1-overexpressing strain 17.24 16.27 0.69 1.09 0.80 0.41 0.42 1.93 ERO1-overexpressing strain 18.98 16.93 0.80 2.41 1.82 0.49 1.02 8.68

(21) The values of the concentrations multiples of the metabolites according to the cultures of the transformed, modified yeast strains prepared above are shown in Table 4 below.

(22) TABLE-US-00004 TABLE 4 Strain Squalene 2,3-oxidosqualene Protopanaxadiol Control 1 1 1 CPR5-overexpressing 1.48 0.59 4.93 strain PDI1-overexpressing 0.97 0.59 1.15 strain ERO1-overexpressing 2.14 0.72 5.19 strain

(23) In Tables 3 and 4, the control group represents the modified PPD yeast strain (S. cerevisiae CEN.PK2-1D Δtrp1::tHMG1+P.sub.GPD1 PgSE+Δleu2::P.sub.GPD1 PgDDS+P.sub.GPD1 PgPPDS+P.sub.GPD1 PgCPR); the CPR5-enhanced strain represents PPD-CPR5(P.sub.CPR5::P.sub.PGK1); the PDI1-enhanced strain represents PPD-PDI1(P.sub.PDI1::P.sub.PGK1); and the ERO1-enhanced strain represents PPD-ERO1(P.sub.ERO1::P.sub.PGK1).

(24) The values in Table 4 indicate multiple values of the metabolites produced from each of the modified yeast strains which had been prepared, wherein the value of the concentration of each of the metabolites (e.g., squalene, 2,3-oxidosqualene, and protopnanxadiol) is set to 1.

(25) Based on the results above, it was confirmed that the transformation of the modified yeast strains had no significant effect on the cell growth. In addition, as a result of the measurement of the concentrations of the intracellular metabolites, it was confirmed that overexpression of CPR5, PDI1, or ERO1 increases the protein folding, thereby increasing the concentrations of the metabolites of ginsenoside biosynthesis through a decrease in the unfolded protein response (UPR). Therefore, based on these results, it can be predicted that the ginsenoside-producing ability is finally improved.